Project Summary The primary goals of this proposal are to develop improved methods for probing the structure and dynamics of RNAs by nuclear magnetic resonance (NMR) spectroscopy and the application of NMR and other biophysical methods to better understand the molecular mechanisms that RNAs use to carry out their varied cellular functions. Our lab studies a number of RNA systems that include the RNA aptamer drug Macugen and newly discovered RNA regulatory elements called riboswitches. Aim 1 will develop improved methods for NMR solution structure determinations of RNA. One focus will be the development of novel alignment techniques for measurement of residual dipolar couplings (RDCs). These RDCs provide long-range structural information that is critical for the determination of the more extended structures adopted by many RNAs. General chemistries for introducing paramagnetic tags into RNAs will be developed and used to obtain RDCs that improve both the global and local structure. In addition, recently developed methods for rapid acquisition of high-resolution four-dimensional NMR will be applied to resonance assignments of RNA. Aim 2 will study the molecular mechanism of inhibition of Macugen for its physiological target, vascular endothelial growth factor (VEGF). Macugen was recently approved by the FDA for treatment of the wet form of age-related macular degeneration, the leading cause of blindness in the elderly. Macugen binds with high affinity and specificity to VEGF, thus blocking the binding of VEGF to cell surface receptors. Multi-dimensional heteronuclear NMR will be used to determine the solution structures of Macugen bound to full-length VEGF and of Macugen bound to the heparin-binding domain of VEGF. These structural studies will be complemented with biochemical studies of VEGF mutants to elucidate the specific interactions that lead to the high affinity of the VEGFMacugen complex. Aim 3 will employ fluorescence resonance energy transfer (FRET) and NMR techniques to study the kinetics and thermodynamics of conformational changes in the secondary and tertiary structures of RNAs. Single molecule FRET will be used to probe transitions between conformational states for both the ligand binding domain and the expression platform of the S-adenosylmethionine riboswitch. These studies will be complemented by NMR experiments that probe the dynamics of the RNA. Overall this proposal will lead to a better understanding of molecular mechanisms that RNAs use to recognize their protein partners and to switch between their functional states.